CN114774531A - 一种用于在细胞内原位检测microRNA的生物传感器 - Google Patents
一种用于在细胞内原位检测microRNA的生物传感器 Download PDFInfo
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Abstract
本发明提供一种用于在细胞内原位检测microRNA的生物传感器，是合成二氧化硅纳米粒子‑羧基聚乙二醇MSN‑PEG‑COOH，将用于检测目的microRNA的发卡探针适配体CPA和MSN‑PEG‑COOH溶解在5×SSC溶液中，在黑暗条件下进行组装反应，反应后离心得到生物传感器；再用透明质酸涂层生物传感器。本发明提供了一种可用于非编码核酸的无酶自组装生物传感器，本发明的传感器对非编码核酸的检测限可达1fM；可用于心肌梗死重要生物表面分子mir‑124的原位检测。本发明的传感器也可以设计成对应不同非编码核核酸的生物传感器，用于疾病的体内原位分析。
Description
技术领域
本发明属于疾病诊断制品制备技术领域，具体涉及一种用于在细胞内原位检测microRNA的生物传感器。
背景技术
MicroRNA是一种长度约为22个核苷酸的非编码小RNA分子序列，可有效调节基因表达。MicroRNA的不适当表达在多种疾病的预后和发病机制中发挥作用。近年来，许多研究人员证明，心血管疾病的发生和发展伴随着特定microRNA表达的变化。因此，microRNA作为生物标志物在心血管疾病的诊断中具有重要意义。
急性心肌梗死(AMI)可诱导大量心肌细胞凋亡，随后的缺血再灌注和氧化应激可诱导多种凋亡相关基因、蛋白和通路发生变化。进一步扩大心肌细胞凋亡，从而加重心血管疾病并增加死亡率。研究表明，microRNAs在氧化应激和心肌梗死的过程中起着关键的调节作用。
最新研究发现，急性心肌梗死发生时miR-124水平显著升高，miR-124在所有吸烟个体中均有强烈表达，并与单细胞改变引起的亚临床动脉硬化风险相关表型增加相关。随机临床试验表明，血清miR-124水平可能是心脏骤停后结果的有用预后指标。miR-124在急性冠状动脉综合征患者中高度表达，以区分需要紧急冠状动脉血管闭塞的脑膜炎。
尽管临床和临床前研究表明miR-124可能在心血管***中发挥关键作用，但传统的microRNA定量方法具有成本高、耗时长、灵敏度低的局限性。为了克服传统方法的局限性，研究人员开发了其他检测的策略，例如传感器和生物传感器，包括比色法、电化学、表面等离子共振、表面增强拉曼光谱(SERS)、电化学发光和荧光技术测量micro RNA。在这些技术中，荧光生物传感器因其成本低、响应时间快、操作简单、适用于生物医学等优点而被更广泛地用于检测microRNA。但已有的生物传感器在进行细胞内原位检测microRNA时效果并不好。
发明内容
本发明的目的是提供一种用于在细胞内原位检测microRNA的生物传感器，从而弥补现有技术的不足。
本发明所提供的用于在细胞内原位检测microRNA的生物传感器，其制备方法如下：
1)合成二氧化硅纳米粒子-羧基聚乙二醇MSN-PEG-COOH
将多面体羟基化二氧化硅纳米粒子MSN-OH用3-氨丙基三乙氧基硅烷(APTES)进行胺化处理，然后用戊二醛溶液进一步羰基化处理，最后与羧基-PEG-胺反应合成MSN-PEG-COOH；
2)组装生物传感器
将用于检测目的microRNA的发卡探针适配体Capture probe adapter(CPA)和MSN-PEG-COOH溶解在5×SSC溶液(pH 7.0)中，在黑暗条件下进行组装反应，反应后离心得到生物传感器；
3)透明质酸涂层生物传感器
先用超声波溶解生物传感器，加入N-乙基-N'-(3-二甲氨基丙基)碳二亚胺(EDC)和N-羟基磺基琥珀酰亚胺钠盐(磺基-NHS)，再加入透明质酸，避光反应；反应结束后，产物用冰乙醇沉淀，透析后冻干，得到透明质酸包封的生物传感器。
本发明再一个方面提供上述的生物传感器的一种用途，是在制备用于原位检测microRNA的制品中的应用。
所述原位检测，是用于在心肌细胞中检测miR-124。
本发明提供了一种可用于非编码核酸的无酶自组装生物传感器，本发明的传感器对非编码核酸的检测限可达1fM；可用于心肌梗死重要生物表面分子mir-124的原位检测。本发明的传感器也可以设计成对应不同非编码核酸的生物传感器，用于疾病的体内原位分析。
附图说明
图1：探针材料形态的TEM表征图；
图2:核酸适配体纳米生物传感器组装结果及RNA稳定性分析图，
图3:纳米生物传感器检测miRNA miR-124的敏感性和特异性分析图；
图4:不同时间条件下H2O2处理后细胞中miR-124表达水平的变化图；
图5：细胞内纳米生物传感器原位检测结果图。
具体实施方式
本发明提供的生物传感器可在细胞内原位检测microRNA，在实施例中记载了在典型的心肌梗死细胞模型中原位检测microRNA-124的效果，但本发明所提供的生物传感器还可以用于在不同的细胞中检测其它的microRNA。
本发明实施例中使用的药品的信息如下：
十六烷基三甲基溴化铵(CTAB)和原硅酸四乙酯(TEOS)等化合物购自AladdinReagent Company。3-氨基丙基三乙氧基硅烷(APTES)、N-乙基-N’-(3-二甲基氨基丙基)碳二亚胺(EDC)、焦碳酸二乙酯、氰基硼氢化钠和N-羟基磺基琥珀酰亚胺钠盐(磺基-NHS)购自Sigma-Aldrich。戊二醛、硅油和NH2-PEG-COOH(MW＝2000)购自Macklin Inc。用于RT-PCR的各种试剂购自TaKaRa。所有实验均使用Milli-Q水(电阻率18.2MΩcm)。合成的miRNA靶点、胺化寡核苷酸CPA和荧光基团标记的寡核苷酸(表1)由北京六合华大基因科技有限公司定制。
表1：本发明使用的寡核苷酸的序列信息表
下面结合实施例和附图对本发明进行详细的描述。
实施例1：合成用于细胞内原位检测microRNA的生物传感器
细胞内原位检测microRNA的生物传感器，其制备方法如下：
1)合成MSN-PEG-COOH
将多面体羟基锡酸盐MSN-OH用3-氨丙基三乙氧基硅烷(APTES)进行胺化处理，将MSN-OH加入到APTES的乙醇溶液中缓慢摇动2～6小时，反应完成后用无水乙醇清洗样品三次，放入110度烘箱干燥10～16小时。然后用戊二醛溶液进一步羰基化处理，上上一步的反应产物加入到戊二醛溶液中缓慢摇动反应2～6小时，反应完成后用水清洗样品三次，室温真空干燥6～12小时。最后与羧基-PEG-胺反应合成MSN-PEG-COOH；上一步的反应产物加入到羧基-聚乙二醇-氨基溶液中缓慢摇动反应6～12小时，反应完成后用水清洗样品三次，室温真空干燥6～12小时。
2)组装生物传感器
将Capture probe adapter(CPA)，本实施例中的CPA为HPN CP-124-dabcyl；和MSN-PEG-COOH溶解在添加有5×柠檬酸钠盐(SSC)溶液(pH7.0)中，在黑暗条件下进行组装反应，反应后离心得到生物传感器；
3)透明质酸涂层生物传感器
先用超声波溶解生物传感器，加入一定量的N-乙基-N'-(3-二甲氨基丙基)碳二亚胺(EDC)和N-羟基磺基琥珀酰亚胺钠盐(磺基-NHS)，反应30分钟，然后加入透明胺化酸，避光反应12～36小时；反应结束后，产物用过量的冰乙醇沉淀，透析后冻干，得到透明质酸包封的生物传感器。
实施例2：生物传感器的理化性质检测
1、探针表征
用透射电镜对封装前后的生物传感器的形貌进行了表征，如图1所示。从图中可以看出，生物传感器的尺寸约为110nm，，而且透明质酸对生物传感器的形态影响不大(图1，图2)。
2、生物传感器和核酸组装表征和RNA稳定性研究
在将特定microRNA检测适配体与生物传感器混合后，通过琼脂糖凝胶实验测试了适配体的状况。结果如图2a和图2b所示。结果表明适配体组装在生物传感器上并保留在琼脂糖凝胶的加载孔中。同时，将miRNA与生物传感器混合，加入一定体积的培养基(DMEM、1640等)在二氧化碳培养箱中孵育过夜，并设立miRNA对照组和生物传感器组作为对照组。取过夜样本进行逆转录后，进行荧光定量PCR实验，测试生物传感器是否能有效保护miRNA不被降解。结果显示在下面的图2c中。结果显示生物传感器可以保护miRNA不被降解。起到稳定miRNA的作用。
3、生物传感器检测范围和错配分析
为了更准确地确定待检测miRNA的浓度，首先使用生物传感器材料对不同浓度的miRNA进行检测，并制作了检测标准曲线，
1)建立目标microRNA检测曲线
准备1fM到100μM不同浓度梯度的目标microRNAs(表1中的miR-124、124-m1、124-m2、124-m3 microRNA中的一种或多种)，并将其溶解在5×SSC中。将10μL的目标样品添加到90μL生物传感器中。混合孵育30min后，用1×SSC洗涤3次，然后用5％乙醇洗涤一次。最后，用100μL 1×SSC重悬，并用全功能微孔板检测器进行测试。每个浓度样品平行检测5次，制作标准曲线。
2)检测目标序列错配
准备浓度为100nM的不同miRNA(表1)miR-124-mis1、miR-124-mis2等，并溶解在5×SSC中。将10μL的miRNA样品添加到90μL生物传感器中，并在室温下反应30分钟。1×SSC洗涤3次，然后用5％乙醇洗涤一次。最后，用100μL的1×SSC重悬，并用全功能微孔板测试仪进行测试。每个样品平行测试5次，比较测试结果。
如图3a所示。从结果可以看出，本发明的生物传感器可用于miRNA的检测，在1fM～100μM浓度范围内与生物传感器的荧光强度呈一定比例关系，检测限为1fM(图3a)。与检测方法相比，本发明的生物传感器的灵敏度更高。
此外，测试了其他几种常见的miRNA和miR-124错配，测试结果如图3b所示。从结果可以看出，在众多浓度为100nM的miRNA中，本发明的生物传感器对待检测的目标miR-124具有特别高的特异性，但对其他miRNA的检测不敏感。这也说明了本发明的生物传感器材料检测的特异性和准确性。
实施例3：生物传感器的细胞内原位检测
1、细胞培养
使用心肌细胞系H9C2细胞系制备了氧化应激心肌细胞梗塞模型以证实我们的实验结果。H9C2细胞系来源于Holistic Gold细胞库(中国北京)。H9C2细胞在DMEM(Sigma)和10％FBS(Gibco)中培养。H9C2与95％空气和5％二氧化碳在37℃下孵育。在无血清培养基中饥饿12小时后，分别用200uM过氧化氢0小时、2小时、4小时、6小时、8小时、10小时制备氧化应激模型。同时，过氧化氢处理8小时后，加入生物传感器，孵育6小时进行检测。
2、氧化应激心肌细胞梗死模型
用H2O2对心肌细胞H9C2进行氧化应激处理，构建心肌梗死模型。用H2O2处理不同时间条件后，收集细胞提取总RNA，然后通过荧光定量PCR分析细胞中miR-124表达水平的变化。从图4的结果可以看出，在H2O2的诱导下，心肌细胞中miR-124的表达持续升高。
3、细胞内原位检测miR-124
使用本发明制备的生物传感器来检测由氧化应激制备的心肌梗塞模型。将纳米生物传感器引入细胞6小时后，用PBS洗涤细胞5次，然后加入4％甲醛的PBS溶液，室温固定细胞10分钟。之后，去除甲醛溶液并用PBS洗涤，用DAPI对细胞进行染色。最后用荧光显微镜进行成像观察，如图5所示。从结果可以看出，在正常心肌细胞中，由于miR-124的低表达，在荧光显微镜下几乎看不到生物传感器的荧光；图5a为H2O2处理后的细胞加入标有FAM的核酸适配子的结果图，图5b为正常细胞加入标有FAM核酸适配子的纳米传感器的结果图，图5c为H2O2处理后的细胞加入纳米传感器结果图，图5d为H2O2处理后的细胞加入标有FAM的核酸适配子的纳米传感器结果图。结果表明纳米传感器可以将标有FAM的核酸适配子运输进细胞内并对特定miRNA进行成像分析。(Scale bar,50μm)。
但在氧化应激心肌梗死模型中，由于生物传感器中miR-124的过表达增强了生物传感器的荧光，可以清楚地看到荧光。类似地，如果将适体直接添加到氧化应激心肌细胞梗塞模型5a中，则适配体无法进入细胞内，从能起到检测特定microRNA-miR-124的作用。
Claims (7)
1.一种用于在细胞内原位检测microRNA的生物传感器，其特征在于，所述的生物传感器的制备方法如下：
1)合成二氧化硅纳米粒子-羧基聚乙二醇MSN-PEG-COOH
将多面体羟基化二氧化硅纳米粒子MSN-OH用3-氨丙基三乙氧基硅烷APTES进行胺化处理，然后用戊二醛溶液进一步羰基化处理，最后与羧基-PEG-胺反应合成MSN-PEG-COOH；
2)组装生物传感器
将用于检测目的microRNA的发卡探针适配体和MSN-PEG-COOH溶解在溶液中，在黑暗条件下进行组装反应，反应后离心得到生物传感器；
3)透明质酸涂层生物传感器
先用超声波溶解生物传感器，加入N-乙基-N'-(3-二甲氨基丙基)碳二亚胺(EDC)和N-羟基磺基琥珀酰亚胺钠盐，再加入透明质酸，避光反应；反应结束后，产物用冰乙醇沉淀，透析后冻干，得到透明质酸包封的生物传感器。
2.如权利要求1所述的生物传感器，其特征在于，所述的用于检测目的microRNA的发卡探针适配体，其中microRNA为miR-124。
3.如权利要求1所述的生物传感器，其特征在于，所述的2)中的溶液为5XSSC溶液，其pH值为7.0。
4.权利要求1所述的生物传感器在制备用于原位检测microRNA的制品中的应用。
5.如权利要求4所述的应用，其特征在于，所述的原位检测microRNA，是在心肌细胞中检测microRNA。
6.如权利要求5所述的应用，其特征在于，所述的microRNA为miR-124。
7.一种用于在心肌细胞中检测microRNA的制品，其特征在于，所述的制品包含有权利要求1所述的生物传感器。
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